Direct affinity measurement of human igg1 binding multimeric antigens

ABSTRACT

Herein is reported a method for determining the binding affinity of the binding sites of a bivalent full length antibody of the human IgG1 subclass to a homo-multimeric antigen comprising the steps of i) incubating a mixture comprising the antibody and a polypeptide that is derived from lysine-gingipain of porphyromonas gingivalis at a pH of from pH 7.5 to pH 8.5, in the presence of a reducing agent, at a temperature of from 30° C. to 42° C., for time of from 10 min. to 240 min. to cleave the antibody into Fabs and Fc-region, and ii) determining the binding affinity of the Fabs of the antibody for its antigen using a surface plasmon resonance method by directly applying the incubated reaction mixture obtained in the previous step in the surface plasmon resonance method and therewith determining the binding affinity of the binding sites of the bivalent full length antibody of the human IgG1 subclass.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/EP2016/079756, having an international filing date of Dec. 5,2016, the entire contents of which are incorporated herein by reference,and which claims benefit under 35 U.S.C. § 119 to European PatentApplication No. 15198556.1, filed on Dec. 9, 2015.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 4, 2018 isnamed P33225-US_Sequence_Listing.txt and is 27,995 bytes in size.

FIELD OF THE INVENTION

Herein is reported a fast and easy method for determining affinityconstants of human IgG1s binding di- or multimeric antigens usingsurface plasmon resonance. The method is based on a highly specific andquantitative digestion with lysine-gingipain of porphyromonas gingivalisgenerating a homogenous pool of intact Fab and Fc-fragments without anyover-digestion of the fragments typically associated with otherproteolytic enzymes.

BACKGROUND OF THE INVENTION

The quality of a biopharmaceutical product is of decisive importance inaddition to its action. Therefore in addition to a detailedinvestigation of the modes of action, it is absolutely essential todetermine the identity, purity and activity of protein-based drugs inorder to use them safely as therapeutic agents.

Although mAbs can be successfully analyzed by means of variousseparation and testing techniques, it has for a long time been difficultto apply and optimize RP-HPLC methods (RP-HPLC, Reversed Phase-HighPerformance Liquid Chromatography) to separate antibody species.However, various modifications of the antibody are often presentsimultaneously during the course of a degradation process, which makesit more difficult to analyze the diverse chromatographic andelectrophoretic bands. Analysis by means of liquid chromatographicseparation methods coupled with high resolution mass spectrometers(LC/MS, liquid chromatography/mass spectrometry) yields informationabout the exact mass of the various species and thus facilitates theidentification of the antibody variants (Dillon, T. M., et al., J.Chromatogr. A, 1053 (2004) 299-305).

Papain, a cysteine protease, cleaves peptide bonds relativelynon-specifically after arginine (R), lysine (K), glutamic acid (E),histidine (H), glycine (G) and tyrosine (Y). If the incubation period issufficiently long, the papain digestion leads to a total hydrolysis.However, antibodies can be cleaved relatively selectively in their hingeregion by a limited proteolysis (Lottspeich, F., and Engels, J. W.,“Bioanalytik Spektrum Akademischer Verlag” Munich 2nd Edition (2006)201-214). The cleavage occurs on the N-terminal side of the disulfidebridges which connect the two heavy chains together. The disulfidebridges are retained in this process so that three fragments (2 Fabfragments, 1 Fc fragment) are obtained after the digestion. The twoN-terminal fragments are referred to as antigen-binding fragments (Fab,antigen-binding fragment), the C-terminal fragment is referred to as thecrystalline fragment (Fc, crystallizing fragment). Each Fab fragment iscomposed of a complete light chain and the amino-terminal half of theheavy chain. The Fc fragment is composed of the two carboxy-terminalhalves of the heavy chains which are still linked together by thedisulfide bridge.

In recent years different IgG specific proteases have been identified.

In WO 2015/40125 streptococcal erythrogenic toxin B (SpeB) is reported.It is described as a cysteine protease from streptococcus pyogenes,shown to cleave IgG in the hinge region into two stable monomeric Fabfragments and one Fc fragment. It is further reported that SpeB cleavesthe hinge region of IgG between positions 238 and 239 according to theKabat numbering system (positions 225 and 226 according to EU numberingsystem).

The cysteine endoprotease IdeS (Immunoglobulin degrading enzyme S) fromthe human pathogen Streptococcus pyogenes which is also referred to asMac-1 or sib-38, is a cysteine protease that specifically cleaves theheavy chain of antibodies of the immunoglobulin G type (IgG). IgG ishitherto the only known substrate of IdeS (Vincents, B., et al.,Biochem. 43 (2004) 15540-15549). IdeS consists of 339 amino acidsincluding a signal peptide comprising 29 amino acids (vonPawel-Rammingen, U., et al., EMBO J. 21 (2002) 1607-1615) where an RGDmotif is formed by the amino acids 214 to 216. IdeS cleaves human IgG(class G immunoglobulin) in the hinge region between positions 249 and250 according to the Kabat numbering system (positions 236 and 237according to EU numbering system) (Gly-Gly) which are contained in therecognition sequence LLGGP. Human IgG2 is cleaved between the aminoacids alanine and glycine in the recognition motif PVAGP. Murineantibodies of the IgG2a and IgG3 type are also cleaved (Vincents, B., etal., Biochem. 43 (2004) 15540-15549).

Porphyromonas gingivalis is a major pathogenic factor of the progressiveperiodontal disease (see e.g. Kadowaki, T., et al., J. Biol. Chem. 269(1994) 21371-21378). Therefrom different enzymes have been isolated,amongst them gingipains, trypsin-like cysteine proteases.

Kikuchi, Y., et al. reported the determination of concentration andbinding affinity of antibody fragments by use of surface plasmonresonance (J. Biosci. Bioeng. 100, (2005) 311-317).

In WO 95/11298 a substantially pure Lys-gingipain complex preparation isprovided, wherein Lys-gingipain being characterized as having anapparent molecular mass of 105 kDa as estimated by sodium dodecylsulfate polyacrylamide gel electrophoresis, where sample is preparedwithout boiling, said Lys-gingipain having amidolytic and proteolyticactivity for cleavage after lysine residues and having no amidolyticand/or proteolytic activity for cleavage after arginine residues,wherein the amidolytic and/or proteolytic activity is inhibited by TLCK,cysteine protease group-specific inhibitors including iodoacetamide andiodoacetic acid, wherein the amidolytic and/or proteolytic activity ofsaid Lys-gingipain is not sensitive to inhibition by leupeptin,antipain, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, serineprotease group-specific inhibitors including diisopropylfluorophosphateand phenylmethyl sulfonylfluoride, and antibodies specific for theLys-gingipain protein complex and its catalytic component, methods forpreparation.

Inagaki, S., et al. reported about antibody responses of periodontitispatients to gingipains of porphyromonas gingivalis (J. Periodont. 74(2003) 1432-1439).

Nguyen, H., et al. reported that surface plasmon resonance is aversatile technique for biosensor applications (Sensors 15 (2015)10481-10510).

SUMMARY OF THE INVENTION

Herein is reported a method for the determination of the bindingaffinity of a full length antibody of the human IgG1 subclass to itsantigen using the fragment antigen binding (Fab) generated thereof in asurface plasmon resonance method whereby the Fabs are generatedenzymatically by incubation with lysine-gingipain of porphyromonasgingivalis and the reaction mixture is directly used for affinitydetermination without intermediate purification.

One aspect as reported herein is a method for determining the bindingaffinity of the binding sites of a bivalent full length antibody of thehuman IgG1 subclass to its homo-multimeric antigen comprising thefollowing steps:

-   -   incubating a mixture comprising the antibody and a polypeptide        that is derived from lysine-gingipain of porphyromonas        gingivalis under conditions and for a time sufficient to cleave        the antibody into Fabs and Fc-region, and    -   determining the binding affinity of the Fabs of the antibody for        their antigen using a surface plasmon resonance method by        directly applying the incubated reaction mixture obtained in the        previous step in the surface plasmon resonance method and    -   thereby determining the binding affinity of the binding sites of        the bivalent full length antibody of the human IgG1 subclass.

One aspect as reported herein is a method for determining the bindingaffinity of the binding sites of a bivalent full length antibody of thehuman IgG1 subclass to a homo-multimeric antigen comprising thefollowing steps:

-   -   incubating a mixture comprising the antibody and        lysine-gingipain of porphyromonas gingivalis or a enzymatically        active fragment thereof at a pH of (from pH) 7.5 to (pH) 8.5, in        the presence of a reducing agent, at a temperature of (from)        30° C. to 42° C., for a time of (from) 10 min. to 240 min to        cleave the antibody into Fabs and Fc-region, and    -   determining the binding affinity of the Fabs of the antibody for        its antigen using surface plasmon resonance by directly applying        the incubated reaction mixture obtained in the previous step in        the surface plasmon resonance method and    -   thereby determining the binding affinity of the binding sites of        the bivalent full length antibody of the human IgG1 subclass.

In one embodiment the method comprises the following steps:

-   -   incubating a mixture comprising the antibody and a polypeptide        that is derived from lysine-gingipain of porphyromonas        gingivalis at a pH of (from pH) 7.5 to (pH) 8.5, in the presence        of a reducing agent, at a temperature of (from) 30° C. to 42°        C., for time of (from) 10 min. to 240 min. to cleave the        antibody into Fabs and Fc-region, and    -   determining the binding affinity of the Fabs of the antibody for        their antigen using a surface plasmon resonance method by        directly applying the incubated reaction mixture obtained in the        previous step in the surface plasmon resonance method and    -   thereby determining the binding affinity of the binding sites of        the bivalent full length antibody of the human IgG1 subclass.

In one embodiment the polypeptide that is derived from lysine-gingipainof porphyromonas gingivalis is the lysine-gingipain of porphyromonasgingivalis. In one embodiment the polypeptide that is derived fromlysine-gingipain of porphyromonas gingivalis comprises the amino acidsequence of SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or afunctional variant thereof. In one embodiment the lysine-gingipain ofporphyromonas gingivalis has the amino acid sequence of SEQ ID NO: 02 orSEQ ID NO: 03 or SEQ ID NO: 04 or is a functional variant thereof. Inone embodiment the polypeptide that is derived from lysine-gingipain ofporphyromonas gingivalis has an amino acid sequence that comprises atleast residues 230 to 739 of SEQ ID NO: 01.

In one embodiment the reducing agent is selected from the groupconsisting of 2-mercaptoethanol, cysteine, and dithiothreitol. In oneembodiment the reducing agent is cysteine. In one embodiment thereducing agent is cysteine at a concentration of (from) 0.5 mM to 10 mM.In one embodiment the reducing agent is cysteine at a concentration ofabout 2 mM.

In one embodiment the pH value is about pH 8.

In one embodiment the temperature is of (from) 35° C. to 38° C. In oneembodiment the temperature is about 37° C.

In one embodiment the incubating is for a time of (from) 30 min. to 120min. In one embodiment the incubating is for a time of about 60 min.

In one embodiment the antibody comprises in the Fc-region the mutationsP329G, L234A and L235A in both heavy chain polypeptides.

One aspect as reported herein is a method for selecting an antibody(specifically binding to a homo-multimeric antigen) comprising thefollowing steps:

-   -   providing a plurality of bivalent full length antibodies of the        human IgG1 subclass binding to the same antigen,    -   determining the binding affinity of each of the antibodies of        the plurality of antibodies to its homo-multimeric antigen with        a method according to any one of claims 1 to 11, and    -   selecting one or more antibodies based on the binding affinity        determined in the previous step.

DETAILED DESCRIPTION OF THE INVENTION Definitions:

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, MD (1991) and is referred to as“numbering according to Kabat” herein. Specifically, the Kabat numberingsystem (see pages 647-660) of Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, MD (1991) is used for the light chainconstant domain CL of kappa and lambda isotype, and the Kabat EU indexnumbering system (see pages 661-723) is used for the constant heavychain domains (CH1, Hinge, CH2 and CH3, which is herein furtherclarified by referring to “numbering according to Kabat EU index” inthis case).

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably.

The term “about” denotes a range of +/−20% of the thereafter followingnumerical value. In one embodiment the term about denotes a range of+/−10% of the thereafter following numerical value. In one embodimentthe term about denotes a range of +/−5% of the thereafter followingnumerical value.

The term “lysine-gingipain of porphyromonas gingivalis” denotes apolypeptide that specifically cleaves human IgG1 and IgG3 subclass heavychains between positions 238 and 239 according to the Kabat numberingsystem (positions 225 and 226 according to EU numbering system), i.e.the hinge region amino acid sequence DKTHTCPPCPAPELLGGPSVF (SEQ ID NO:05) is cleaved after the second amino acid residue resulting in thefragments DK (SEQ ID NO: 06) and THTCPPCPAPELLGGPSVF (SEQ ID NO: 07). Inone embodiment the polypeptide, i.e. the lysine-gingipain ofporphyromonas gingivalis, comprises the amino acid sequence of SEQ IDNO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or a functional variantthereof. In one embodiment the polypeptide, i.e. the lysine-gingipain ofporphyromonas gingivalis, has an amino acid sequence that comprises atleast residues 230 to 739 of SEQ ID NO: 01. The “lysine-gingipain ofporphyromonas gingivalis” has the EC number 3.4.22.47 and is alsodenoted as gingipain K, KGP, Lys-gingipain, PrtP proteinase,lysine-specific cysteine protease, lysine-specific gingipain,lysine-specific gingipain K, or lysine-specific gingipain proteinase.The full length amino acid sequence of an exemplary lysine-gingipain ofporphyromonas gingivalis is denoted in SEQ ID NO: 01. This polypeptideis an endopeptidase with strict specificity for lysyl bonds. Theenzymatic activity of the polypeptide is activated by the addition ofabout 30 2-mercaptoethanol, about 50 mM cysteine, about 30 mMdithiothreitol, about 2 mM EDTA, about 2 mM EGTA or glutathione. It isactive in the pH range from pH 6.5 to pH 9.5, with a pH of from about pH7.5 to about pH 8.5 (preferably about pH 8.0) being suitable for thehydrolysis of immunoglobulins. In an exemplary IgG degradation methodthe following conditions are used: IgG (final concentration 15 μM), KGP(final concentration 10 nM active protease), Tris buffer (0.1 mol/L, pH8.0), EDTA (final concentration 1 mM), L-cysteine (final concentration 2mM), 37° C. Human IgGs are cleaved once but if the glycostructures areremoved a second cleavage might occur. The enzymatic cleavage can benegatively affected if chaotropic reagents and/or detergents arepresent. Thus, in one embodiment the method is performed in the absenceof chaotropic reagents and/or detergents from all solutions used in themethod.

The term “full-length antibody” denotes an antibody which comprises twoso called light immunoglobulin chain polypeptides (light chain) and twoso called heavy immunoglobulin chain polypeptides (heavy chain). Each ofthe heavy and light immunoglobulin chain polypeptides of a full-lengthantibody contains a variable domain (variable region) (generally theamino terminal portion of the polypeptide chain) comprising bindingregions that are able to interact with an antigen. Each of the heavy andlight immunoglobulin chain polypeptides of full-length antibodycomprises a constant region (generally the carboxyl terminal portion).The constant region of the heavy chain mediates the binding of theantibody i) to cells bearing a Fc gamma receptor (FcγR), such asphagocytic cells, or ii) to cells bearing the neonatal Fc receptor(FcRn) also known as Brambell receptor. It also mediates the binding tosome factors including factors of the classical complement system suchas component (C1q). The variable domain of an antibody's light or heavychain in turn comprises different segments, i.e. four framework regions(FR) and three hypervariable regions (CDR).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal are highly specific, being directed against a singleantigenic site. Furthermore, in contrast to polyclonal antibodypreparations, which include different antibodies directed againstdifferent antigenic sites (determinants or epitopes), each monoclonalantibody is directed against a single antigenic site on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they may be synthesized uncontaminated by otherantibodies. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies and is not to be construed as requiring production of theantibody by any particular method.

The “Fc-region” of an antibody is not involved directly in binding tothe antibody's antigen, but exhibits various effector functions.Depending on the amino acid sequence of the constant region of the heavychains, antibodies (immunoglobulins) are divided in the classes: IgA,IgD, IgE, IgG, and IgM. Some of these classes are further divided intosubclasses (isotypes), i.e. IgG in IgG1, IgG2, IgG3, and IgG4, or IgA inIgAl and IgA2. According to the immunoglobulin class to which anantibody belongs are the heavy chain constant regions of immunoglobulinsare called □□□ IgA), □ (IgD), □ (IgE) □□□ (IgG), and □ (IgM),respectively. The antibodies according to the invention belongpreferably to the IgG class. An “Fc-region of an antibody” is a termwell known to the skilled artisan and defined on basis of the papaincleavage of antibodies.

Affinity Determination:

Normally antibodies, such as full length antibodies of the IgG class,are bivalent. Thus, when determining the affinity it should be a “true”affinity avoiding the avidity effect of the bivalent binder. Todetermine the affinity and binding kinetics of antibodies binding bi- ormultivalent targets it is therefore necessary to turn the bivalentantibodies into monovalent binding entities like fragmentantigen-binding (Fab) units.

Currently methods for the determination of the affinity of bivalentantibodies are two step methods:

-   -   1: cleavage of the antibody to be analyzed to generate        monovalent binding entities, and    -   2: purification of the reaction mixture of 1.

Alternatively it is possible to re-clone and express the Fab fragmentfor which even more time and labor are required as for the approachoutlined above.

Generation Of IgG-Fragments:

Often, Fab fragments are generated by partial proteolytic digestions ofIgGs with unspecific proteases like papain or pepsin, which cleave aboveor below the hinge region, respectively. The fragments contain thedisulphide bonds that join the heavy chains, but the cleavage is belowthe site of the disulphide bond between the light chain and heavy chain(Porter, 1959; Nisonoff et al., 1960; Akita and Nakai, 1993, Andrew andTitus, 2003; Mage, 1987; Zhao et al., 2009; Andrew, S. M. and J. A.Titus. 2003).

IgGs digested with pepsin results in F(ab′)2 fragments that aresubsequently mildly reduced to give Fab′ fragments. Most likely thehinge region is more susceptible to the attack of proteases as it isexposed and flexible. Subsequently, the fragments are then purified fromthe digestion mix.

However, lack of reproducibility, uncut IgG and over-digestion is oftena problem.

With papain digestion, e.g., it is difficult to obtain homogeneous Fabs(Parham, 1983; 1986; Mage, 1987). Immobilized papain products (e.g.papain agarose resins; see e.g. Tischer, W., and V. Kasche, 1999; Luo,Q., et al. 2002) allow better control of the digestion reaction andefficient removal of the Fab and Fc fragments from the crude proteasedigest; nevertheless purification is still required.

Another approach to obtain monomeric antigen-binding fragments includethe generation of F(ab′)2 fragments by digestion with ImmunoglobulinG-degrading enzyme of S. pyogenes (IdeS) and mild reduction with2-mercaptoethylamine (2-MEA) to generate Fab′ fragments (vonPawel-Rammingen, U., et al. 2002+2003; Ishikawa, E. and S. Yoshitake,1980; DeSilva, B. S., et al., 1995).

The two antigen-binding domains of an antibody of the IgG class can alsobe obtained by reducing the IgG to two half-IgGs (rIgG; see e.g. Billah,M. M., et al., 2010). It is the product of selectively reducing just thehinge-region disulphide bonds which are the most accessible and easiestto reduce, especially with a mild reducing agents like 2-MEA.

Finally, a Fab can also be obtained by recombinant expression of thelight chain and the heavy chain Fd-fragment (VH-CH1) (Zhao et al.,2009). This is however time consuming and laborious if several differentFabs are needed for e.g. a comparison.

A limited digestion using the endoproteinase Lys-C in a 40 min digestionof hIgG1's to analyze the chain assembly by mass spectrometry has beenreported in PCT/EP2015/057164. Using this procedure Lys-C exclusivelycuts once above the hinge region generating Fab and Fc-fragments.

A protease that cleaves selectively in the upper hinge region ofantibodies of the IgGs class is streptococcal erythrogenic toxin B(SpeB) from S. pyogenes (von Pawel-Rammingen, U., et al. 2002). Thisprotease requires reducing agent like DTT or TCEP in the range of 1-5 mMfor activity (Persson, H., et al., 2013; www.genovis.com/fabulous)resulting in the concomitant reduction of the interchain thiols of thedigested antibody.

IgG-specific proteases and their cleavage sites are shown in thefollowing Table (see also Brerski, R. J. and Jordan, R. E., mAbs 2(2010) 212-220).

protease specificity recognition sequence fragments plasminDK↓THTCPPCPAPELLGGPSVF 2xFab (SEQ ID NO: 05) 1xFc lysine-gingipain ofhuman DK↓THTCPPCPAPELLGGPSVF 2xFab porphyromonas IgG1 and 1xFcgingivalis IgG3, IgA human neutrophil DKT↓HTCPPCPAPELLGGPSVF 2xFabelastase 1xFc papain IgG, specific DKTH↓TCPPCPAPELLGGPSVF 2xFab only in1xFc limited proteolysis streptococcal DKTHT↓CPPCPAPELLGGPSVF 2xFcerythrogenic toxin 2xLC B (SpeB) from 1xFc S. pyogenes glutamylDKTHTCPPCPAPE↓LLGGPSVF 1xF(ab′)2 endopeptidase I from S. aureus,Cathepsin G pepsin IgG1 > IgG2 DKTHTCPPCPAPEL↓LGGPSVF 1xF(ab′)2 multipleHC-Fc fragments Immunoglobulin DKTHTCPPCPAPELLG↓GPSVF 1xF(ab′)2G-degrading 2xHC-Fc enzyme of S. pyogenes (IdeS)

The P. gingivalis proteases have been studied since more than 30 years.They have been identified as cysteine-proteinases requiring the presenceof reducing agents for activity. One of them is the cysteine proteasegingipain K (EC. 3.4.22.47).

Scott et al. purified lysine-gingipain of porphyromonas gingivalis (KGP)back in 1993 (Scott, C. F., et al., J. Biol. Chem. 268 (1993)7935-7942).

Scott et al. identified cysteine, dithiothreitol, glutathione and2-mercaptoethanol to be suitable reducing agents for the activation ofKGP.

KGP cleaves peptides with Lys in the P1 position, and the residue at P2appears to be less important. However, if P2 is occupied by Lys or Arg,hydrolysis appears to be blocked. KGP is capable of hydrolyzing proteinsubstrates such as BSA, casein, hemoglobin, acid-soluble human placentaltype I collagen, human IgG, and IgA (Curtis, M. A., et al., Crit. Rev.Oral Biol. Med. 12 (2001) 192-216).

The amino acid sequence of lysine-gingipain of porphyromonas gingivalisincluding an identification of the respective domains was reported byOkamoto, K., et al. (J. Biochem. 120 (1996) 398-406). The kgp gene wasreported and deposited by Slakeski, N., et al. under accession numberU75366 and AAB60809.1 (Oral Microbiol. Immunol. 14 (1999) 92-97).Several C-terminally truncated but active forms have been identified. Ithas been found that for the C-terminally truncated proteinsKGP(Δ1292-1732), KGP(Δ1157-1732), KGP(Δ738-1732), KGP(Δ681-1732) andKGP(Δ602-1732) enzymatic activity was only barely measurable for thelast two mutants (see e.g. Sztukowska, M., et al., Mol. Microbiol. 54(2004) 1393-1408).

KGP has a narrow specificity for synthetic substrates, limited topeptide bonds containing arginine and lysine residues, respectively, butthey can nevertheless degrade immunoglobulins G and A in a limiteddegradation manner (Yamamoto, K., et al., In: Proteases: newPerspectives (1999), V. Turk (ed.), Birkhäuser Verlag Basel (CH),175-184; Yamamoto, K., et al., In: N Katunuma, H Kido, H Fritz, J Travis(Eds): Medical Aspects of Proteases and Protease Inhibitors. 105 Press,Amsterdam, 139-149; Kadowaki, T., et al., J. Biol. Chem. 269 (1994)21371-21378; Abe, N., et al., J. Biochem. 123 (1998) 305-312).

Comparative properties of envelope-associated arginine-gingipains (RGP)and lysine-gingipain (KGP) of porphyromonas gingivalis have beenreported in 1998 by Fujimura et al. (Microbiol. Lett. 163 (1998)173-179). The enzymes were commonly activated by reducing reagents suchas mercaptoethanol, dithiothreitol and cysteine. RGP-B was activatedmarkedly by glycyl-glycine and KGP was activated significantly by EDTAand EGTA. The hydrolytic activities of RGPs and KGP to chromogenicsynthetic substrates were limited to the compounds with arginine andlysine in the P-1 positions, respectively. When IgG was treated with thethree enzymes separately, it was demonstrated that two new fragments of34 kDa and 15 kDa (SDS under reducing conditions) were generated in eachreaction product. The optimum pH for the activity of KGP was found to be7.5. Thiol reagents activated both RGPs and KGP, whereas dithiothreitolwas the best activator of KGP (at 20-30 mM), followed by mercaptoethanol(at 20-30 mM) and cysteine (at more than 1.5 mM but less than 10 mM).KGP split only X-Y-Lys-pNA.

Vincents, B., et al. reported that gingipain K of porphyromonasgingivalis can hydrolyze subclass 1 and 3 of human IgG, whereby theheavy chain of IgG1 was cleaved at a single site within the hingeregion, generating Fab and Fc fragments and that IgG3 was also cleavedwithin the heavy chain, but at several sites around the CH2 region(FASEB J., 25 (2011) 3741-3750). Cleavage of IgG2 is not mediated by KGP(Guentsch, A., et al., J. Periodont. res. 48 (2013) 458-465).

An high-resolution crystal structure of KGP active site was reported byde Diego, I., et al. suggesting that catalysis may require a catalytictriad, Cys477-His444-Asp388, rather than the cysteine-histidine dyadnormally found in cysteine peptidases (J. Biol. Chem. 289 (2014)32291-32302).

Different antibody fragments are described in the following:

-   -   the F(ab′)2 fragment:    -   the F(ab′)2 fragment has a molecular weight of about 110 kDa and        comprises the two antigen-binding site of a full length antibody        of the IgG class connected via the hinge-region disulfide bonds;        it is void of most, but not all, of the Fc-region    -   Fab′ fragment:    -   the Fab′ fragment has a molecular weight of about 55 kDa; it can        be formed by the reduction of the hinge-region disulfide bonds        of a F(ab′)2 fragment; the Fab′ fragment comprises a free        sulfhydryl group; as it is derived from F(ab′)2 it may contain a        small portion of the Fc.-region    -   fragment antigen binding—Fab:    -   the Fab has a molecular weight of about 50 kDa; it is a        monovalent binding fragment that can be obtained from antibodies        of the IgG and IgM class; it comprises the VH and CH1 domains of        the heavy chain and a complete light chain both linked by an        intramolecular disulfide bond    -   Fv fragment:

the Fv fragment has a molecular weight of about 25 kDa; it is thesmallest antibody fragment that contains a complete antigen-binding site(VH domain and VL domain);

-   -   the VH and VL domains of the Fv fragment are held together by        non-covalent interactions    -   “rIgG” fragment:    -   the “rIgG” fragment denotes a half-antibody that is obtained by        reducing just the hinge-region disulfide bonds of a full length        antibody (e.g. using 2-MEA); it has a molecular weight of about        75 kDa    -   fragment crystallizable—Fc-fragment:    -   the Fc-fragment has a molecular weight of about 50 kDa; it        comprises the CH2 and CH3 domains of the heavy chain of a full        length antibody and part of the hinge region; the two chains are        held together by one or more disulfide bonds (in the hinge        region); the Fc-fragment cannot bind the antigen, but it is        responsible for the effector functions of the full length        antibody.

The Method as Reported Herein:

Different from antibodies binding monomeric antigens, it is not feasibleto determine the affinity without the influence of avidity of antibodiesbinding di- or multimeric antigens, for example by surface plasmonresonance (SPR).

Affinity describes the strength of a single interaction between antibodyand its antigen. A bivalent antibody of the IgG class has twoantigen-binding sites, and the avidity is commonly applied to antibodyinteractions in which multiple antigen-binding sites simultaneouslyinteract with the target antigen, often in multimeric structures. Theavidity of an antibody refers to the accumulated strength of multipleaffinities. Avidity is commonly obtained regarding interactions in whichmultiple antigen-binding sites, often in multimeric structures, areinvolved. To determine the affinity of antibodies it is necessary toconvert the bivalent antibodies into monovalent binding entities likeantigen-binding fragments (Fab).

The determination of affinity constants of antibodies targeting di- ormultimeric antigens by SPR therefore necessitates the generation ofmonomeric antigen-binding fragments, such as e.g. Fabs. The generationof Fabs is however laborious. Often, Fabs are generated by partialproteolytic digestions with papain.

Herein is reported a fast and easy method for the generation of Fabsfrom full length antibodies of the IgG1 subclass. It has been found thatthe lysine-gingipain of porphyromonas gingivalis can be used for thegeneration of Fabs from full length antibodies of the IgG1 subclass aswith this enzyme a highly specific and quantitative protease digestiongenerating a homogenous pool of intact Fab and Fc-fragments without anyover-digestion typically associated with other proteolytic enzymes canbe achieved and the reaction mixture can directly, i.e. without anyintermediate purification, be applied to a surface plasmon resonancechip.

In more detail this is done by in solution digestion and direct kineticaffinity determination of the Fab fragment by SPR without any priorpurification or cleaning step. The complete digestion by thelysine-gingipain of porphyromonas gingivalis of human IgG1s was verifiedby ESI-QTOF-MS.

The method as reported herein can be used for the determination ofkinetic rate constants of human or humanized antibodies, e.g. of thesubclass IgG1 or comprising an Fc-region derived from the human subclassIgG1, specifically binding to di- or multimeric antigens using a surfaceplasmon resonance method. The method comprises in one embodiment thefollowing steps: 1) incubating the antibody with the lysine-gingipain ofporphyromonas gingivalis to cleave it completely generating a homogenouspool of Fabs and Fc-fragments, and 2) determining the binding affinityof the Fab in the digestion mixture by surface plasmon resonance (SPR).Direct SPR on the digestion mixture allows precise kineticcharacterization of the Fab fragment without any prior purification.

It has been found that the affinity constants determined by SPR of Fabsof antibodies of the IgG1 subclass obtained by digesting with thelysine-gingipain of porphyromonas gingivalis without subsequentpurification correspond to affinity constants of Fabs obtained byrecombinant expression, or by digesting with papain and subsequentpurification prior to SPR measurement.

One aspect as reported herein is a method for determining the bindingaffinity of the binding sites of a bivalent full length antibody of thehuman IgG1 subclass to its antigen comprising the following steps:

-   -   incubating a mixture comprising the antibody and a polypeptide        that is derived from lysine-gingipain of porphyromonas        gingivalis under conditions and for a time sufficient to cleave        the antibody into Fabs and Fc-region, and    -   determining the binding affinity of the Fabs of the antibody for        their antigen using a surface plasmon resonance method by        directly applying the incubated reaction mixture obtained in the        previous step in the surface plasmon resonance method and    -   thereby determining the binding affinity of the binding sites of        the bivalent full length antibody of the human IgG1 subclass.

In one embodiment the method comprises the following steps:

-   -   incubating a mixture comprising the antibody and a polypeptide        that is derived from lysine-gingipain of porphyromonas        gingivalis at a pH of from pH 7.5 to pH 8.5, in the presence of        a reducing agent, at a temperature of from 30° C. to 42° C., for        time of from 10 min. to 240 min. to cleave the antibody into        Fabs and Fc-region, and    -   determining the binding affinity of the Fabs of the antibody for        their antigen using a surface plasmon resonance method by        directly applying the incubated reaction mixture obtained in the        previous step in the surface plasmon resonance method and    -   thereby determining the binding affinity of the binding sites of        the bivalent full length antibody of the human IgG1 subclass.

In one embodiment the polypeptide that is derived from lysine-gingipainof porphyromonas gingivalis is the lysine-gingipain of porphyromonasgingivalis. In one embodiment the polypeptide that is derived fromlysine-gingipain of porphyromonas gingivalis comprises the amino acidsequence of SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or afunctional variant thereof. In one embodiment the polypeptide that isderived from lysine-gingipain of porphyromonas gingivalis has an aminoacid sequence that comprises at least residues 230 to 739 of SEQ ID NO:01.

In one embodiment the reducing agent is selected from the groupconsisting of 2-mercaptoethanol, cysteine, and dithiothreitol. In oneembodiment the reducing agent is cysteine. In one embodiment thereducing agent is cysteine at a concentration of from 0.5 mM to 10 mM.In one embodiment the reducing agent is cysteine at a concentration ofabout 2 mM.

In one embodiment the pH value is about pH 8.

In one embodiment the temperature is of from 35° C. to 38° C. In oneembodiment the temperature is about 37° C.

In one embodiment the incubating is for a time of from 30 min. to 120min. In one embodiment the incubating is for a time of about 60 min.

In one embodiment the antibody comprises in the Fc-region the mutationsP329G, L234A and L235A in both heavy chain polypeptides.

In one embodiment the antigen is multimeric antigen. In one embodimentthe antigen is a homo-multimeric antigen. In one embodiment the antigenis selected from the group consisting of vascular endothelial growthfactor A (VEGF-A), carcinoembryonic antigen (CEA), angiopoietin-2(ANG2), and fibroblast activation protein (FAP).

One aspect as reported herein is a method for selecting an antibodycomprising the following steps:

-   -   providing a plurality of bivalent full length antibodies of the        human IgG1 subclass binding to the same antigen,    -   determining the binding affinity of each of the antibodies of        the plurality of antibodies to its antigen with a method        according to any one of claims 1 to 11, and    -   selecting one or more antibodies based on the binding affinity        determined in the previous step.

The method as reported herein allows for a fast determination of theaffinity of a bivalent full length antibody for its antigen without therequirements to recombinantly produce a single binding site version ofthe antibody. With the method as reported herein the determination ofthe biding affinity of the bivalent full length antibody for its antigenis possible without the need for an intermediate purification of thereaction mixture that has been used for the generation of the Fabs ofthe bivalent full length antibody.

The method as reported herein has been exemplified in the following withthe antibody bevacizumab. Bevacizumab is a humanized anti-VEGF antibodyof the human IgG1 subclass. The therapeutic antibody bevacizumab binds adimeric antigen, i.e. VEGF-A (dimeric is a form of homo-multimeric).

The quality of the bevacizumab Fabs and digests was analyzed by UHRESI-QTOF mass spectrometry. The deconvoluted mass spectra of thepurified Fab following a papain digest, and a purified recombinant Fabprovided proof for the high quality of both materials as only the massesof the Fab fragments could be detected.

In more detail, complete digestion of bevacizumab by thelysine-gingipain of porphyromonas gingivalis was verified byelectrospray ionization mass spectrometry after desalting of thereaction mixture using a size exclusion chromatography. No fragmentationor side products could be identified in the MS spectra.

For comparison bevacizumab has been digested using the enzyme papain.Form the MS spectra it can be seen that papain is not suitable forfunctional assessment due to unspecific fragmentation of the antibodyand loss of function.

The respective MS-spectra are shown in FIG. 1 (one hour digestion withthe lysine-gingipain of porphyromonas gingivalis), FIG. 2 (1.5 hoursdigestion with papain), and FIG. 3 (2 hour digestion with papain). Itcan be seen that no antibody fragmentation beside the single cleavage inthe hinge region occurred when the lysine-gingipain of porphyromonasgingivalis was used.

In more detail, the quality of the recombinant bevacizumab Fab andpapain and lysine-gingipain of porphyromonas gingivalis digests wereanalyzed by UHR ESI-QTOF mass spectrometry. The deconvoluted massspectra of the purified Fab following a papain digest, and a purifiedrecombinant Fab revealed the high quality of both materials as only themasses of the intact Fab 48208 Da (theoretical average mass: 48208 Da)and 47726 Da (theoretical average mass: 47726), respectively, could bedetected. The evaluation of the mass spectrum of bevacizumab digestedwith papain revealed not only the presence of the 48207 Da Fab(theoretical average mass: 48208 Da) and the Fc-fragments (multiplemasses present due to heterogeneity of the Fc N-glycan's). In addition,unassignable fragments corresponding to the masses x:23422 Da and 23453Da, y:34587 Da, and z:47607 Da were detected in the papain digest. Incontrast the deconvoluted mass spectrum of bevacizumab digested with thelysine-gingipain of porphyromonas gingivalis demonstrated only thepresence of the 47969 Da Fab (theoretical average mass: 47970 Da) andthe Fc-fragment (multiple masses present due to the Fc N-glycan's). Thedigestion with the lysine-gingipain of porphyromonas gingivalis wascomplete without any undigested or single cut IgG (IgG without one Fab)detectable by mass spectrometry. Nor could any unspecific digestion,over-digestion, or further degradation of the fragments be detected inthe crude digestion mixture of the lysine-gingipain of porphyromonasgingivalis digest.

The method as reported herein was performed with differentbevacizumab-derived samples:

-   -   1) full length bivalent antibody    -   2) recombinantly produced Fab    -   3) Fab produced with a method as reported herein (without        intermediate purification) (determined directly after the        incubation and after 24 hours additional incubation in the        presence of functional lysine-gingipain of porphyromonas        gingivalis)    -   4) Fab produced with papain (without termination of the reaction        and without intermediate purification)    -   5) Fab produced with papain (with termination of the reaction,        without intermediate purification)    -   6) Fab produced with papain (with intermediate purification)

In order to compare the affinities of the different produced Fabs ofbevacizumab the binding affinities of bevacizumab digested with thelysine-gingipain of porphyromonas gingivalis without purification of theFab and the binding affinities of a recombinant transiently expressedbevacizumab Fab, a purified Fab following a papain digest weredetermined.

For determining the affinities a murine anti-His-tag antibody wasimmobilized and the dimeric VEGF-A conjugated to a His-tag was capturedon the sensor chip surface. Afterwards, the analytes binding to VEGF-Awere injected and flew over the surface. The derived sensorgrams werefitted to a 1:1 Langmuir binding model and used to determine theassociation rate constants ka, the dissociation rate constants kd, andthe binding constants KD. Generally, the rate and binding constants forthe Fab fragments were all very similar (see Table below). The bindingconstant of the Fab in the lysine-gingipain of porphyromonas gingivalisdigestion mixture was found to be 1.1 nM, and those of the recombinantFab and the purified Fab after digestion with papain were determined to0.8 and 1.0 nM, respectively. The KD of the full length bivalentantibody was determined to be 0.18 nM demonstrating the avid binding tothe dimeric VEGF-A. But when the papain digestion mixture was applied tothe immobilized chip surface, we did not observe binding to the captureddimeric VEGF-A. Consequently, no binding constant could be determinedfor the papain digestion mixture. It has been found that the VEGF-Asurface was damaged after applying the papain containing samples as itcould not be used anymore.

The results are presented in the following Table.

ka kd KD sample [1/Ms] [1/s] [nM] full length bevacizumab (avidity)1.61E+05 2.96E−05 0.18 recombinant bevacizumab Fab 9.03E+04 7.37E−05 0.8(affinity) bevacizumab digested lysine-gingipain 5.18E+04 5.83E−05 1.1of porphyromonas gingivalis, without purification (without additionalincubation) bevacizumab digested with papain could not be determined asno (without termination of the reaction and binding signal was observedwithout intermediate purification) bevacizumab digested with papaincould not be determined as no (with termination of the reaction, bindingsignal was observed without intermediate purification) bevacizumabdigested with papain 7.95E+04 8.02E−05 1.0 (with intermediatepurification)

It can be seen that as the lysine-gingipain of porphyromonas gingivalisis specific for human IgG1, it does not destroy the immobilized chipsurface. In contrast thereto no binding was observed after the notpurified papain digestion reaction mixture was applied to theimmobilized chip surface. The VEGF surface could not be used any moreafter applying the papain containing sample as it has been damaged bythe presence of papain.

The respective SPR diagrams are shown in FIG. 4A to 4D.

Storage of the lysine-gingipain of porphyromonas gingivalis-digestedbevacizumab and repeated affinity determinations by SPR allowed toconclude the digests to be stable at 4° C. for at least 24 and 48 hours,respectively, i.e. no further digestion or fragmentation occurred.

Beside the use of lysine-gingipain of porphyromonas gingivalis for thedetermination of affinities of human IgG1s binding di-or multimericantigens, the protease can also be used in cases where IgG1s bindingmonomeric antigens are difficult to immobilize on the SPR metal surface.

In addition, the lysine-gingipain of porphyromonas gingivalis will bevery beneficial for the structural analysis of the Fab fragments andstructure-function relationships of human IgG1-antigen binding at atomicresolution, e.g., by X-ray crystallography. Compared with IgGs, Fabfragments are more amenable to crystallization.

Recombinant Methods:

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an antibody as described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method ofproducing an antibody as reported herein is provided, wherein the methodcomprises culturing a host cell comprising a nucleic acid encoding theantibody, as provided above, under conditions suitable for expression ofthe antibody, and optionally recovering the antibody from the host cell(or host cell culture medium).

For recombinant production of an antibody, nucleic acid encoding anantibody, e.g., as described above, is isolated and inserted into one ormore vectors for further cloning and/or expression in a host cell. Suchnucleic acid may be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.No. 5,648,237, U.S. Pat. No. 5,789,199, and U.S. Pat. No. 5,840,523.(See also Charlton, K. A., In: Methods in Molecular Biology, Vol. 248,Lo, B. K. C. (ed.), Humana Press, Totowa, N.J. (2003), pp. 245-254,describing expression of antibody fragments in E. coli.) Afterexpression, the antibody may be isolated from the bacterial cell pastein a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.No. 5,959,177, U.S. Pat. No. 6,040,498, U.S. Pat. No. 6,420,548, U.S.Pat. No. 7,125,978, and U.S. Pat. No. 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR—CHOcells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

General chromatographic methods are known to a person skilled in the arte.g. Chromatography, 5th edition, Part A: Fundamentals and Techniques,Heftmann, E. (ed.); Elsevier Science Publishing Company, New York,(1992); Advanced Chromatographic and Electromigration Methods inBiosciences, Deyl, Z. (ed.), Elsevier Science B V, Amsterdam, TheNetherlands, (1998); Chromatography Today, Poole, D. F., and Poole, S.K., Elsevier Science Publishing Company, New York, (1991); Scopes,Protein Purification: Principles and Practice (1982); Sambrook, J., etal. (ed.), Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; orCurrent Protocols in Molecular Biology, Ausubel, F. M., et al. (eds.),John Wiley & Sons, Inc., New York.

Citations:

Andrew S. M., Titus J. A., 2003, Fragmentation of immunoglobulin G. CurrProtoc Cell Biol. Unit 16.4. (Chapter 16).

Mage, E. L. M., 1987, Preparation of Fab and F(ab′) fragments frommonoclonal antibodies, p. 79-97. In: L. B. Schook (Ed.), Monoclonalantibody production techniques and applications, Marcel Dekker Inc., NewYork.

Parham, P., J Immunol. 131 (1983) 2895-2902.

Parham, P., 1986, Preparation and purification of active fragments frommouse monoclonal antibodies, p. 14.1-14.23. In: D. M. Weir (Ed.),Handbook of Experimental Immunology, 4th Ed. Blackwell ScientificPublications, Oxford.

Porter, R. R., Biochem J. 73 (1959) 119-126.

Nisonoff, A., et al., Arch. Biochem. Biophys. 89 (1960) 230-244.

Zhao, Y. L., et al., Protein Expr. Purif. 67 (2009) 182-189.

Akita, E. M., and S. Nakai, J. Immunol. Methods 162 (1993) 155-164.

Tischer, W. and V. Kasche, Trends Biotechnol. 17 (1999) 326-335.

Luo, Q., et al., J. Chrom. 776 (2002) 139-147.

von Pawel-Rammingen, U., et al., EMBO J. 21 (2002) 1607-1615.

von Pawel-Rammingen, U. and L. Bjorck, Curr. Opin. Microbiol. 6 (2003)50-55.

Ishikawa, E. and S. Yoshitake, J. Immunol. Methods 38 (1980) 117-123.

DeSilva, B. S. and G. S. Wilson, G. S., J. Immunol. Methods 188 (1995)9-19.

Billah, M. M., E T A L., Bioelectrochem. 80 (2010) 49-54.

Persson, H., E T A L., Infect. Immun. 81 (2013) 2236-2241.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 UHR ESI-QTOF mass spectrometry of bevacizumab digested withlysine-gingipain of porphyromonas gingivalis for one hour at 37° C. OnlyFab and Fc fragments were detected.

FIG. 2 UHR ESI-QTOF mass spectrometry of bevacizumab digested withpapain for 1.5 h at 37° C. Beside Fab and Fc fragments, several Fab- andantibody fragments were detected.

FIG. 3 UHR ESI-QTOF mass spectrometry of bevacizumab digested withpapain for 2 h at 37° C. Several antibody fragments were detected. Faband Fc fragment could not be identified.

FIG. 4A Surface plasmon resonance sensorgrams of bevacizumab.

FIG. 4B Surface plasmon resonance sensorgrams of a recombinantbevacizumab Fab.

FIG. 4C Surface plasmon resonance sensorgrams of bevacizumab digestedwith lysine-gingipain of porphyromonas gingivalis.

FIG. 4D Surface plasmon resonance sensorgrams of bevacizumab,bevacizumab digested with papain without termination of the digest, andbevacizumab digested with papain with termination of the digest.

EXAMPLES

Bevacizumab was obtained from Roche Diagnostics GmbH (Mannheim,Germany). Papain was obtained as suspension with a concentration of 10mg/mL from Sigma-Aldrich/Roche Diagnostics GmbH. Lysine-gingipain ofporphyromonas gingivalis was obtained under the trade name GingisKHANfrom Genovis (Lund, Sweden). GingisKHAN was reconstituted in 200 μLdouble distilled water (ddH2O) resulting in 2000 U/200 μL, and the 10×reducing agent was freshly prepared in 50 μL ddH2O (final concentration:20 mM cysteine) prior to each digestion.

Example 1 Transient Fab Expression and Purification

The antibody light chain and heavy chain Fd-fragments were ordered asgene syntheses and cloned via unique restriction sites using standardcloning procedures into separate expression vectors for each chainenabling secretory expression in HEK cells growing in suspension.Transfection (1:1 plasmid ratios) into HEK293-F cells (Invitrogen, Cat.No. 510029) was performed according to the cell supplier's instructionsusing Maxiprep (Qiagen, Cat. No. 12163) preparations of the antibodyvectors, Opti-MEM I medium (Invitrogen, Cat. No. 31985) 293fectin(Invitrogen, Cat. No. 31985070), and an initial cell density of1-2×10E+06 viable cells/mL in serum-free FreeStyle 293 expression medium(Invitrogen, Cat. No. 12338018). Antibody containing cell culturesupernatants were harvested after 7 days of cultivation in shake flasksby centrifugation at 14,000× g for 30 min. and filtered through a 0.22μm sterile filter (Thermo Scientific, Cat. No. 566-0020). The antibodieswere purified directly from the supernatant, or the supernatant wasstored at −80° C. until purification. The quality of the purified Fabwas analyzed by SEC and BioAnalyzer.

Example 2 Enzymatic Cleavage of Bevacizumab with Papain WithoutPurification:

The antibody was diluted in 20 mM Histidine, 140 mM NaCl, pH 6.0 to afinal concentration of 1 mg/mL, added 2 μL 250 mM L-cysteine(Sigma-Aldrich, Schnelldorf, Germany) and 10.9 μL diluted papain (7.34U/mL in 20 mM Histidine, 140 mM NaCl, pH 6.0), and incubated 1 h at 37°C.

With Purification:

The antibody was incubated with Papain (0.8 U/mg mAb;Sigma-Aldrich/Roche) in presence of 5 mM Cystein for 170 minutes at 37°C. To isolate the Fab from non-cleaved antibodies, Fc-fragments andPapain, the mixture was applied to a CaptureSelect IgG-CH1 andMabSelectSuRe affinity chromatography (GE Healthcare) according tomanufacturer protocol. Finally, a size exclusion chromatography using aSuperdex 75 10/300 GL column (GE Healthcare) was performed using 140 mMNaCl, 20 mM histidine (pH 6.0) as running buffer. Protein concentrationof the Fab was determined by measuring the optical density (OD) at 280nm, using the molar extinction coefficient calculated on the basis ofthe amino acid sequence. The purity was analyzed by SDS-PAGE in thepresence and absence of a reducing agent (5 mM 1,4-dithiotreitol) andstaining with Coomassie brilliant blue.

Example 3 Enzymatic Cleavage of Bevacizumab with Lysine-Gingipain ofPorphyromonas Gingivalis

GingisKHAN was reconstituted in 200 μL ddH2O resulting in 2000 U/200 μL,and the 10× reducing agent was freshly prepared in 50 μL ddH2O (finalconcentration: 20 mM Cysteine) prior to each digestion. 100 μg antibodywas diluted to a final concentration of 1 mg/mL in 100 mM Tris, pH 8.0and subsequently digested with 10 μL GingisKHAN and 11 μL of freshlyprepared 10× reducing agent at 37° C. for 1 hour.

Example 4 ESI-QTOF Mass Spectrometry

Samples were desalted by HPLC on a Sephadex G25 column (Kronlab, 5×250mm, TAC05/250G0-SR) using 40% acetonitrile with 2% formic acid (v/v).The total mass was determined via ESI-QTOF MS on a maXis 4G UHR-QTOF MSsystem (Bruker Daltonik) equipped with a TriVersa NanoMate source(Advion). Calibration was performed with sodium iodide (Waters ToFG2-Sample Kit 2 Part: 700008892-1). For the recombinant and purifiedFabs, data acquisition was done at 900-2600 m/z (ISCID: 0.0 eV), for thehIgG1s or digested hIgG1s, data acquisition was done at 900-4000 m/z(ISCID: 0.0 eV). The raw mass spectra were evaluated and transformedinto individual relative molar masses using an in-house developed Rochesoftware tool. For visualization of the results, the same in-housedeveloped software was used to generate deconvoluted mass spectra.

Example 5 Surface Plasmon Resonance

Binding affinities and kinetics were investigated by surface plasmonresonance using a BlAcore T200 instrument (GE Healthcare). Allexperiments were performed at 25° C. using PBS-T (10 mM Na2HPO4, 140 mMNaCl, 0.05% Tween 20, pH 7.4) as running and dilution buffer. Ananti-His-tag (GE Healthcare, #28995056) or an anti-human Fab antibody(GE Healthcare, #28958325) was immobilized on a Series S CMS Sensor Chip(GE Healthcare, #29104988) using standard amine coupling chemistry.Histidine-tagged human VEGF or full length IgG/Fabs were captured on thesurface leading to a response between 10 and 50 RU. The analytes wereinjected for 180 s at concentrations from 2.2 nM up to 1800 nM onto thesurface (association phase) at a flow rate of 30 μL/min. Thedissociation phase was monitored for up to 3600 sec. by washing withrunning buffer. The surface was regenerated by injecting 10 mM GlycinepH 1.5 for 60 sec. at a flow rate of 5 μL/min. Bulk refractive indexdifferences were corrected by subtracting the response obtained from amock surface and by subtracting blank injections (double referencing).The derived curves were fitted to a 1:1 Langmuir binding model using theBlAevaluation software.

1. A method for determining the binding affinity of the binding sites ofa bivalent full length antibody of the human IgG1 subclass to ahomo-multimeric antigen comprising the following steps: incubating amixture comprising the antibody and lysine-gingipain of porphyromonasgingivalis or an enzymatically active fragment thereof at a pH of 7.5 to8.5, in the presence of a reducing agent, at a temperature of 30° C. to42° C., for a time of 10 min. to 240 min. to cleave the antibody intoFabs and Fc-region, and determining the binding affinity of the Fabs ofthe antibody for its antigen using surface plasmon resonance by directlyapplying the incubated reaction mixture obtained in the previous step inthe surface plasmon resonance method and thereby determining the bindingaffinity of the binding sites of the bivalent full length antibody ofthe human IgG1 subclass.
 2. The method according to claim 1, wherein thelysine-gingipain of porphyromonas gingivalis has the amino acid sequenceof SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or is a functionalvariant thereof.
 3. The method according to claim 2, wherein thepolypeptide that is derived from lysine-gingipain of porphyromonasgingivalis has an amino acid sequence that comprises at least residues230 to 739 of SEQ ID NO:
 01. 4. The method according to claim 1, whereinthe reducing agent is selected from the group consisting of2-mercaptoethanol, cysteine, and dithiothreitol.
 5. The method accordingto claim 4, wherein the reducing agent is cysteine.
 6. The methodaccording to claim 4, wherein the reducing agent is cysteine at aconcentration of 0.5 mM to 10 mM.
 7. The method according to claim 1,wherein the pH value is about pH
 8. 8. The method according to claim 1,wherein the temperature is of 35° C. to 38° C.
 9. The method accordingto claim 1, wherein the incubating is for a time of about 60 min. 10.The method according to claim 1, wherein the antibody comprises in theFc-region the mutations P329G, L234A and L235A in both heavy chainpolypeptides.
 11. A method for selecting an antibody specificallybinding to a homo-multimeric antigen comprising the following steps:providing a plurality of bivalent full length antibodies of the humanIgG1 subclass binding to the same homo-multimeric antigen, determiningthe binding affinity of each of the antibodies of the plurality ofantibodies to its antigen with a method according to any one of claims 1to 10, and selecting one or more antibodies based on the bindingaffinity determined in the previous step.
 12. The method according toclaim 1, wherein the incubated mixture is used for the determination ofthe binding affinity without intermediate purification.